Vaccine for protection against Lawsonia intracellularis

ABSTRACT

The present invention pertains to the use of a non-live carbohydrate containing composition, the carbohydrate being also found in live  Lawsonia intracellularis  cells in association with the outer cell membrane of these cells, for the manufacture of a vaccine for protection against an infection with  Lawsonia intracellularis , the vaccine being in a form suitable for systemic administration.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of co-pending U.S.application Ser. No. 13/657,210, filed Oct. 22, 2012, which has nowissued as U.S. Pat. No. 8,597,662, which is a divisional of U.S.application Ser. No. 12/988,083, filed Oct. 15, 2010, now Abandoned,which is the National filing under 35 U.S.C. §371 of PCT/EP2009/054516,filed Apr. 16, 2009, which claims priority under 35 U.S.C. §119(e) toU.S. provisional Application No. 61/046,161, filed Apr. 18, 2008 andU.S. provisional Application No. 61/111,756 filed Nov. 6, 2008, andclaims foreign priority under 35 U.S.C. §119(a) to European ApplicationNo. 08154764.8, filed Apr. 18, 2008, and European Application No.081054738.2, filed Nov. 6, 2008. The contents of PCT/EP2009/054516 andU.S. application Ser. Nos. 12/988,083 and 13/657,210 are herebyincorporated by reference in their entireties.

The present invention pertains to a vaccine for protection against aninfection with Lawsonia intracellularis, a vaccine in this sense being acomposition that at least provides a decrease in a negative influence ofthe infection with Lawsonia intracellularis, such negative influencebeing e.g. tissue damage and/or clincal signs such as decreased weightgain, diarrhea, etc.

Proliferative enteropathy (also called enteritis or ileitis) in manyanimals, in particular pigs, presents a clinical sign and pathologicalsyndrome with mucosal hyperplasia of immature crypt epithelial cells,primarily in the terminal ileum. Other sites of the intestines that canbe affected include the jejunum, caecum and colon. Weanling and youngadult pigs are principally affected with typical clinical manifestationof rapid weight loss and dehydration. Natural clinical disease in pigsoccurs worldwide. The disease is consistently associated with thepresence of intracellular curved bacteria, presently known as Lawsoniaintracellularis.

In general, oral vaccination against Lawsonia intracellularis has shownto be an economically efficient measure to control Ileitis and to allowa better exploitation of the genetic growth potential of the pig(Porcine Proliferative Enteropathy Technical manual 3.0, July 2006;available from Boehringer Ingelheim). Furthermore, oral rather thanparenteral vaccination will reduce the transmission of blood-borneinfections such as PRRS via multi-use needles and the reduction ofinjection site reactions and needles retained in carcasses. It willreduce animal and human stresses, time, labour costs and effort comparedto individual vaccination (McOrist: “Ileitis—One Pathogen, SeveralDiseases” at the IPVS Ileitis Symposium in Hamburg, Jun. 28, 2004).

It is generally understood that the advantage of an attenuated livevaccine approach is that the efficacy of immunity is usually relativelygood, as the host's immune system is exposed to all the antigenicproperties of the organism in a more “natural” manner. Specifically forintracellular bacterial agents such as Lawsonia intracellularis, thelive attenuated vaccine approach is believed to offer the best availableprotection for vaccinated animals, due to a full and appropriate T cellbased immune response. This is in contrast with the variable to poorimmunity associated with subunit or killed vaccine types forintracellular bacteria. This is also specifically true for obligateintracellular bacteria such as Lawsonia intracellularis or the Chlamydiasp, which cause pathogenic infections within the mucosa. Studiesindicate that whole live attenuated forms of the intracellular bacteriain question are best delivered to the target mucosa, that they arerequired as whole live bacterial forms to produce a fully protectiveimmune response in the target mucosa but also that they areimmunologically superior compared to use of partial bacterialcomponents.

It has become a general understanding that a vaccine against Lawsoniaintracellularis needs to be administered orally (see i.a. TechnicalManual 3.0 as referred to here-above). This is based on the fact thatthe basis of the body's resistance to Ileitis is the local immunity inthe intestine, which is the product of cell-mediated immunity and localdefense via antibodies, especially IgA. According to current knowledge,serum antibodies (IgG) do not give any protection simply because they donot reach the gut lumen. It has been demonstrated in studies that oralvaccination produces cell-mediated immunity as well as local productionof IgA in the intestine (Murtaugh, in Agrar- and Veterinär-Akademie,Nutztierpraxis Aktuell, Ausgabe 9, Juni 2004; and Hyland et al. inVeterinary Immunology and Immunopathology 102 (2004) 329-338). Incontrast, intramuscular administration did not lead to protection.Moreover, next to the general understanding that a successful vaccineagainst intracellular bacteria has to induce cell-mediated immunity aswell as the production of local antibodies, the skilled practitionerknows that only a very low percentage of orally ingested antigens areactually absorbed by the enterocytes, and that the incorporation ofLawsonia intracellularis into the cell is an active process initiated bythe bacterium. Accordingly an inactivated vaccine would provide theintestine with insufficient immunogenic antigen (Haesebrouck et al. inVeterinary Microbiology 100 (2004) 255-268). This is why it is believedthat only attenuated live vaccines induce sufficient cell-mediatedprotection in the intestinal cells (see Technical Manual 3.0 as referredto here-above). At present there is only one vaccine on the market toprotect against Lawsonia intracellularis, viz. Enterisol® Ileitismarketed by Boehringer Ingelheim. This vaccine is a live vaccine fororal administration indeed.

It is an object of the present invention to provide an alternativevaccine to protect against an infection with Lawsonia intracellularis.To this end it has been devised to use a non-live carbohydratecontaining composition, the carbohydrate being also found in liveLawsonia intracellularis cells in association with the outer cellmembrane of these cells, for the manufacture of a vaccine for protectionagainst an infection with Lawsonia intracellularis, the vaccine being ina form suitable for systemic administration. Surprisingly, against thepersistent general understanding how to combat Lawsonia intracellularis,it was found that by using a carbohydrate containing non-livecomposition, for example extracted from the outer cell membrane ofLawsonia intracellularis, as an antigen in a vaccine, one can induce aprotection against Lawsonia intracellularis that is comparable with oreven improved with respect to the protection provided by using the livevaccine Enterisol® Ileitis (administered according to the correspondinginstructions), when the antigen is administered systemically, i.e. in away that it reaches the circulatory system of the body (comprising thecardiovascular and lymphatic system), thus affecting the body as a wholerather than a specific locus such as the gastro-intestinal tract.Systemic administration can be performed e.g. by administering theantigens into muscle tissue (intramuscular), into the dermis(intradermal), underneath the skin (subcutaneous), underneath the mucosa(submucosal), in the veins (intravenous) etc. Apart from the very goodprotection obtainable, an important advantage of the present non-livevaccine is that it is an inherent safety when compared to a livevaccine.

In general, the carbohydrate containing composition can be used tomanufacture a vaccine by using art-known methods that basically compriseadmixing the antigenic carbohydrate containing composition (or acomposition derived therefrom, such as a dilution or concentrate of theoriginal composition or an extract, one or more purified componentsetc.) with a pharmaceutically acceptable carrier, e.g. a liquid carriersuch as (optionally buffered) water or a solid carrier such as commonlyused to obtain freeze-dried vaccines. As such, manufacturing can takeplace in an industrial environment but also, the antigens could be mixedwith the other vaccine constituents in situ (i.e. at a veterinaries', afarm etc.), e.g. (immediately) preceding the actual administration to ananimal. In the vaccine, the antigens should be present in animmunologically effective amount, i.e. in an amount capable ofstimulating the immune system of the target animal sufficiently to atleast reduce the negative effects of a post-vaccination challenge withwild-type micro-organisms. Optionally other substances such asadjuvants, stabilisers, viscosity modifiers or other components areadded depending on the intended use or required properties of thevaccine. For systemic vaccination many forms are suitable, in particularliquid formulations (with dissolved, emulsified or suspended antigens)but also solid formulations such as implants or an intermediate formsuch as a solid carrier for the antigen suspended in a liquid. Systemicvaccination, in particular parenteral vaccination (i.e. not trough thealimentary canal), and suitable (physical) forms of vaccines forsystemic vaccination have been known for more than 200 years.

It is noted that subunits of Lawsonia intracellularis cells have beenreported as antigens in a vaccine for protection against this bacterium.However, these are mainly recombinant proteins and hitherto none of themhas proven to be able and provide good protection. Killed bacteria(which inherently contain the carbohydrate that is also found in liveLawsonia intracellularis cells in association with the outer cellmembrane) are also suggested as antigens in vaccines against Lawsoniaintracellularis but no vaccines based on killed whole cells haveactually been tested and reported to provide good protection. Apart fromthat, systemic administration has not been used in conjunction withthese killed bacteria, because of the general understanding that thereis no reasonable expectation of success for systemic administration ofantigens to locally (i.e. in the intestines) combat Lawsoniaintracellularis.

In this respect it is noted that in WO 97/20050 (Daratech PTY Ltd)mentions the use of killed Lawsonia intracellularis bacteria to immunizepigs. However, systemic administration is not mentioned. Based on thecurrent knowledge that vaccination is only effective upon oraladministration, it is commonly understood that the oral route was theadministration route chosen for the experiments described in theDaratech application. Another patent application that mentions killedbacteria is WO 2005/011731 (Boehringer Ingelheim). However, actuallydisclosed is only the use of a live vaccine administered orally. It isnot shown that a killed vaccine may be effective, let alone that thekilled vaccine can be given systemically. EP 843 818 (BoehringerIngelheim) describes the intramuscular administration of a killedvaccine (paragraph [0115] in combination with paragraph [0119]). In par[0115] it is stated that the bacteria were killed by storing them at 4°C. at normal atmospheric conditions. As is commonly known however, undersuch conditions Lawsonia intracellularis bacteria survive. Thus, thisdocument does not teach the subject matter of the present invention. Itis also noted that a carbohydrate containing composition, wherein thecarbohydrate is also found in live Lawsonia intracellularis cells inassociation with the outer cell membrane of these cells, is known fromKroll et al. (Clinical and Diagnostic Laboratory Immunology, June 2005,693-699). However, this composition is used for diagnostics. It has notbeen tested as a protective antigen for reasons as stated here-above.

In an embodiment, the carbohydrate containing composition is materialresulting from the killing of Lawsonia intracellularis bacteria. It hasbeen found that a very convenient way of providing the carbohydrate foruse according to the present invention is to simply kill Lawsoniaintracellularis cells and use the material resulting from that as asource for the carbohydrate. To extract the carbohydrate from livingcells could in theory also be done (analogous to the creation of livingghost cells by removing the cell wall) but requires more sophisticatedand thus more expensive techniques. The material as a whole could beused, e.g. a suspension of whole cells or a lysate of Lawsoniaintracellularis cells, or one could purify or even isolate thecarbohydrate out of the material. This method can be performed by usingrelatively simple art-known techniques.

In a preferred embodiment the carbohydrate containing compositioncontains whole cells of killed Lawsonia intracellularis bacteria Thishas proven to be the most convenient way to provide the carbohydrate asan antigen in the vaccine. Besides, the efficacy of the vaccine is evenfurther increased, possibly since this way of offering the antigen tothe immune system of the target animal better mimics the naturalenvironment of the carbohydrate.

In an embodiment the vaccine comprises an oil in water adjuvantcontaining oil droplets of sub-micrometer size. In general, an adjuvantis a non-specific immunostimulating agent. In principal, each substancethat is able to favor or amplify a particular process in the cascade ofimmunological events, ultimately leading to a better immunologicalresponse (i.e. the integrated bodily response to an antigen, inparticular one mediated by lymphocytes and typically involvingrecognition of antigens by specific antibodies or previously sensitizedlymphocytes), can be defined as an adjuvant. It has been shown thatusing an oil in water adjuvant containing oil droplets of sub-micrometersize provides a very good protection against Lawsonia intracellularis.Indeed, the application of oil in water adjuvants as such is common inconnection with non-live antigens. However, it is generally known thatthe best immunostimulating properties are obtained when the oil dropletsare large in diameter. In particular, oil droplets with a diameterbeneath 1 micrometer are in particular used when it is believed thatsafety is an important issue. In that case, one could use small dropletssince these are known to evoke less tissue damage, clinical signs etc.However, in the case of obtaining protection for a gut associateddisorder via systemic vaccination (as is the case in the presentinvention), one would choose large droplets since one would expect thatthe immune response has to be boosted significantly. In contrast, wefound that using small oil droplets in the composition provided verygood results with respect to protection against Lawsoniaintracellularis.

In an even preferred embodiment, the adjuvant comprises droplets ofbiodegradable oil and droplets of mineral oil, the droplets ofbiodegradable oil having an average size that differs from the averagesize of the droplets of mineral oil. It has been shown that the use of amixture of biodegradable oil and mineral oil provides very good resultswith regard to efficacy and safety. In addition to this, stability ofthe composition is very high, which is an important economic advantage.The stability has proven to be very good, in particular when the average(volume weighed) size of either the biodegradable oil droplets or themineral droplets is below 500 nm (preferably around 400 nm).

In an embodiment, the vaccine further comprises antigens of Mycoplasmahyopneumoniae and Porcine circo virus. Hitherto combination vaccines ofLawsonia intracellularis have been suggested in the prior art. However,not many of such combinations have actually been tested for efficacy.The reason for this is that it is generally understood that combinationof antigens with antigens of Lawsonia intracellularis can only lead tosuccessful protection if the Lawsonia antigens are provided as live(attenuated) cells. In this respect, we refer to WO 2005/011731, whichalso suggests all kinds of combination vaccines based on Lawsoniaintracellularis. However, regarding the description and claim structurethe patent application, the assignee (Boehringer Ingelheim) appears tobe convinced that combination vaccines are only expected to have areasonable chance of success when the Lawsonia antigens are present inthe form of live cells. The same is true for WO2006/099561, alsoassigned to Boehringer Ingelheim. Indeed, based on the common generalknowledge this is an obvious thought.

The invention will be further explained using the following examples.

Example 1 describes a method to obtain a substantially protein freecarbohydrate containing composition and a vaccine that is made by usingthis composition.

Example 2 describes an experiment wherein a second vaccine according tothe present invention is compared with the vaccine currently on themarket and an experimental vaccine comprising subunit proteins ofLawsonia intracellularis.

Example 3 describes an experiment wherein two different vaccinesaccording to the present invention are compared with the vaccinecurrently on the market.

Example 4 describes an experiment wherein a dosage affect of a vaccineaccording to the invention is established.

EXAMPLE 1

In this example a method is described to obtain a substantially proteinfree carbohydrate composition associated with the outer cell membrane ofLawsonia intracellularis cells and a vaccine that can be made using thiscomposition. In general, a carbohydrate is an organic compound thatcontains carbon, hydrogen, and oxygen, usually in the ratio 1:2:1.Examples of carbohydrates are sugars (saccharides), starches,celluloses, and gums. Usually they serve as a major energy source in thediet of animals. Lawsonia intracellularis is a gram negative bacterium,which thus contains an outer membrane that is not constructed solely ofphospholipid and protein, but also contains carbohydrates, in particularpolysaccharide (usually polysaccharides such as lipopolysaccharide,lipo-oligosaccharde, or even non-lipo polysaccharides).

Carbohydrate Fraction for Vaccine Preparation

Twenty milliliters of buffered water (0.04 M PBS, phosphate bufferedsaline) containing Lawsonia intracellularis cells at a concentration of3.7E8 (=3.7×10⁸) cells/ml was taken. The cells were lysed by keepingthem at 100° C. for 10 minutes. Proteinase K (10 mg/ml) in 0.04 M PBSwas added to a final concentration of 1.7 mg/ml. This mixture wasincubated at 60° C. for 60 minutes in order to degrade all proteins andkeep the carbohydrates intact. Subsequently, the mixture was incubatedat 100° C. for 10 minutes to inactivate the Proteinase K. The resultingmaterial, which is a carbohydrate containing composition, in particularcontaining the carbohydrates as present in live Lawsonia intracellularisbacteria in association with their outer cell membrane (see paragraphbelow), was stored at 2-8° C. until further use. The composition wasformulated in Diluvac forte adjuvant. This adjuvant (see also EP 0 382271) comprises 7.5 weight percent vitamine E acetate droplets with anaverage volume weighted size of approximately 400 nm, suspended in waterand stabilized with 0.5 weight percent of Tween 80 (polyoxyethylenesorbitan mono-oleate). Each milliliter vaccine contained material thathad been extracted from 1.2E8 Lawsonia intracellularis cells.

Immune Precipitation of Lawsonia Carbohydrate Antigens

Two batches of monoclonal antibodies (MoAb's) raised against whole cellLawsonia intracellularis were precipitated with saturated Na₂SO₄ at roomtemperature according to standard procedures. The precipitate waspelleted by centrifugation (10.000 g for 10 minutes). The pellet waswashed with 20% Na₂SO₄ and resuspended in 0.04 M PBS. Tylosyl activatedDynal beads (DynaBeads, DK) were pre washed with 0.1 M NaPO₄ (pH 7.4),according the manual of the manufacturer. Of each batch of MoAb's 140 μgwas taken and added to 2E8 pre washed beads and incubated overnight at37° C. The beads were pelleted by centrifugation and non-bound MoAb'swere removed by aspiration of the supernatant. Spectrophotometricalmeasurements showed that between 20 and 35% of the added MoAb's hadbound to the beads. Two batches of 1 ml Lawsonia intracellularis cells(3.7E8/ml) in 0.04 M PBS were sonicated for 1 minute. The resulting celllysates were added to the Tylosyl activated beads—monoclonal complexesand incubated overnight at 4° C. The Tylosyl activated beads—monoclonalcomplexes were washed three times with 0.1 M NaPO₄ (pH 7.4). The boundcompounds were eluted by washing the beads in 0.5 ml 8M urea in 0.04 MPBS (E1); 0.5 ml 10 mM Glycine pH 2.5 (E2); and 0.5 ml 50 mM HCl (E3),in a sequential manner. After elution E2 and E3 were neutralized witheither 100 pl and 200 μl 1 M Tris/HCl (pH8.0).

Samples were taken from each step and loaded onto SDS-PAGE gels. Gelswere stained using Commassie Brilliant Blue (CBB) and Silver staining orblotted. The blots were developed using the same MoAb's as mentionedhere-above. Inspection of the gels and blots showed that the MoAb'srecognized bands with an apparent molecular weight of 21 and 24 kDa thatwere not seen on the CBB gels but were visible on de Silver stainedgels. Also, it was established that the fraction of the cells that boundto the MoAb's was Proteinase K resistant. Thus, based on these resultsit can be concluded that this fraction contains carbohydrates (namely:all protein is lysed, and sonified DNA fractions will not show as aclear band in a Silver stain), and that the carbohydrates are inassociation with (i.e. forming part of or being bound to) the outer cellmembrane of Lawsonia intracellularis (namely: the MoAb's raised againstthis fraction also recognized whole Lawsonia intracellularis cells).Given the fact that Lawsonia intracellularis is a gram-negativebacterium, the carbohydrate composition is believed to comprisepolysaccharide(s).

EXAMPLE 2

This experiment was conducted to test a convenient way to formulate thecarbohydrate antigen in a vaccine, viz. via a killed whole cell (alsoknown as bacterin). As controls the commercially available vaccineEnterisol® ileitis and an experimental subunit vaccine comprisingprotein subunits were used. Next to this unvaccinated animals were usedas a control.

Experimental Design of Example 2

An inactivated whole cell vaccine was made as follows. Live Lawsoniaintracellularis cells derived from the intestines of pigs with PPE weregathered. The cells were inactivated with 0.01% BPL(beta-propiolactone). The resulting material, which inherently is anon-live carbohydrate containing composition in the sense of the presentinvention (in particular since it contains the carbohydrates as presentin live Lawsonia intracellularis bacteria in association with theirouter cell membrane), was formulated in Diluvac forte adjuvant (seeExample 1) at a concentration of approximately 2.8×10⁸ cells per mlvaccine.

The subunit vaccine contained recombinant P1/2 and P4 as known from EP1219711 (the 19/21 and 37 kDa proteins respectively), and therecombinant proteins expressed by genes 5074, 4320 and 5464 as describedin W02005/070958. The proteins were formulated in Diluvac forteadjuvant. The vaccine contained approximately 50 pgrams of each proteinsper milliliter.

Forty 6-week-old SPF pigs were used. The pigs were allotted to 4 groupsof ten pigs each. Group 1 was vaccinated once orally (at T=0) with 2 mllive “Enterisol® ileitis” (Boehringer Ingelheim) according to theinstructions of the manufacturer. Group 2 and 3 were vaccinated twiceintramuscularly (at T=0 and T=4w) with 2 ml of the inactivated Lawsoniawhole cell vaccine and the recombinant subunit combination vaccine asdescribed here-above, respectively. Group 4 was left as unvaccinatedcontrol. At T=6w all pigs were challenged orally with homogenized mucosainfected with Lawsonia intracellularis. Subsequently all pigs were dailyobserved for clinical signs of Porcine Proliferative Enteropathy (PPE).At regular times before and after challenge serum blood (for serology)and faeces (for PCR) were sampled from the pigs. At T=9w all pigs wereeuthanized and necropsied. Histological samples of the ileum were takenand examined microscopically.

The challenge inoculum was prepared from infected mucosa: 500 grams ofinfected mucosa (scraped from infected intestines) were mixed with 500ml physiological salt solution. This mixture was homogenized in anomnimixer for one minute at full speed on ice. All pigs were challengedorally with 20 ml challenge inoculum at T=6w.

At T=0, 4, 6, 7, 8 and 9w a faeces sample (gram quantities) and a serumblood sample of each pig was taken and stored frozen until testing. Thefaeces samples were tested in a quantitative PCR (Q-PCR) test andexpressed as the logarithm of the amount found in picograms (pg). Serumsamples were tested in the commonly applied IFT test (immuno fluorescentantibody test to detect antibodies against whole Lawsoniaintracellularis cells in the serum). For histological scoring a relevantsample of the ileum was taken, fixed in 4% buffered formalin, routinelyembedded and cut into slides. These slides were stained withHematoxylin-Eosin (HE stain) and with an immunohistochemical stain usinganti-Lawsonia intracellularis monoclonal antobidies (IHC stain). Theslides were examined microscopically. The histology scores are asfollows:

HE stain:

no abnormalities detected score = 0 doubtful lesion score = ½ mildlesions score = 1 moderate lesions score = 2 severe lesions score = 3

IHC stain:

no L. intracelluaris bacteria evident score = 0 doubtful presence ofbacteria score = ½ presence of single/small numbers of bacteria in theslide score = 1 presence of moderate numbers of bacteria in the slidescore = 2 presence of large numbers of bacteria in the slide score = 3

All data were recorded for each pig individually. The score per groupwas calculated as the mean of the positive animals for the differentparameters after challenge. The non-parametric Mann-Whitney U test wasused to evaluate the statistical significance (tested two-sided andlevel of significance set at 0.05).

Results of Example 2

Serology

Before first vaccination all pigs were seronegative when tested for IFTantibody titres. After vaccination with the whole cell bacterin (group2) pigs developed high IFT antibody titres whereas the controls and thepigs vaccinated with the subunit vaccine remained negative untilchallenge (Table 1). Two of the Enterisol® vaccinated pigs (group 1)developed moderate IFT titres whereas all other pigs in this groupremained seronegative. After challenge all pigs developed high IFTantibody titers. Mean results are depicted in table 1 (with the useddilution, 1.0 was the detection level on the lower side).

TABLE 1 Mean IFT antibody titres (2log) of pig serum after vaccinationand challenge Group T = 0 weeks T = 4 weeks T = 6 weeks T = 9 weeks 1<1.0 1.1 1.7 >11.4 2 <1.0 3.7 >11.8 >12.0 3 <1.0 <1.0 <1.0 >11.6 4 <1.0<1.0 <1.0 >12.0

Real-Time PCR on Faeces Samples

Before challenge all faeces samples were negative. After challengepositive reactions were found in all groups. Group 1 (p=0.02), group 2(p=0.01) and group 3 (p=0.03) had a significantly lower shedding levelcompared to the control. A post-challenge overview is given in table 2.

TABLE 2 Mean results of PCR on faeces samples (log pg) after vaccinationand challenge T = 9 Total post- Group T = 6 weeks T = 7 weeks T = 8weeks weeks challenge 1 0 1.3 3.6 1.8 6.3 2 0 0.8 2.8 1.9 5.5 3 0 0.53.8 2.0 5.9 4 0 0.8 4.9 4.9 10.0

Histology Scores

Group 2 had the lowest histology HE score (p=0.05), IHC score (p=0.08)and total histology score (p=0.08). The other groups had higher scoresand were not significantly different from the control group. See table3.

TABLE 3 Mean histology score for the ileum. Group HE score IHC scoreTotal score 1 1.8 1.5 3.3 2 1.3 1.5 2.7 3 1.8 1.6 3.4 4 2.4 2.3 4.7

Conclusions with Regard to Example 2

From the results it can be concluded that systemic administration of thenon-live whole cell Lawsonia intracellularis vaccine which inherentlycontains the carbohydrate as found also in association with the outermembrane of live Lawsonia intracellularis cells, induced at leastpartial protection. All parameters studied and histology scores weresignificantly or nearly significantly better compared to the controls.

EXAMPLE 3

This experiment was conducted to test a vaccine comprising acarbohydrate containing composition as antigen. A second vaccine to betested contained in addition to killed whole cells of Lawsoniaintracellularis, antigens of Mycoplasma hyopneumoniae and Porcine circovirus (the “combi” vaccine). As a control the commercially availableEnterisol® ileitis vaccine was used. Next to this, unvaccinated animalswere used as a second control.

Experimental Design of Example 3

The vaccine based on a substantially protein free carbohydratecontaining composition was obtained as described under Example 1.

The experimental combi vaccine contained inactivated Lawsoniaintracellularis whole cell antigen (see Example 2 for the used method ofproviding the inactivated bacteria) at a level of 1.7×10⁸ cells/ml. Nextto this it contained inactivated PCV-2 antigen (20 pgrams of the ORF 2encoded protein of PCV 2 per ml; the protein being expressed in a baculovirus expression system as commonly known in the art, e.g. as describedin WO 2007/028823) and inactivated Mycoplasma hyopneumoniae antigen (thesame antigen in the same dose as is known from the commerciallyavailable vaccine Porcilis Mhyo®, obtainable from Intervet, Boxmeer, TheNetherlands). The antigens were formulated in a twin emulsion adjuvant“X”. This adjuvant is a mixture of 5 volume parts of adjuvant “A” and 1volume part of adjuvant “B”. Adjuvant “A” consists of mineral oildroplets with an approximate average (volume weighed) size around 1 μm,stabilised with Tween 80 in water. Adjuvant “A” comprises 25 weight % ofthe mineral oil and 1 weight % of the Tween. Rest is water. Adjuvant “B”consists of droplets of biodegradable vitame E acetate with anapproximate average (volume weighed) size of 400 nm, stabilised alsowith Tween 80. The adjuvant “B” comprises 15 weight % of vitamine Eacetate and 6 weight % of Tween 80, rest is water.

Sixty-four 3-day-old SPF piglets were used. The pigs were allotted tofour groups of 14 piglets and one group of 8 piglets (Group 4). Group 1was vaccinated intramuscularly at 3 days of age with 2 ml of the combivaccine, followed by a second vaccination at 25 days of age. Group 2 wasvaccinated intramuscularly once with 2 ml combi vaccine at 25 days ofage. Group 3 was vaccinated orally with 2 ml Enterisol® ileitis(Boehringer Ingelheim) at 25 days of age according to prescriptions.Group 4 was vaccinated intramusculary at 3 and 25 days of age with 2 mlof the non-protein carbohydrate vaccine. Group 5 was left unvaccinatedas a challenge control group. At 46 days of age all pigs were challengedorally with homogenized infected mucosa. Subsequently all pigs weredaily observed for clinical signs of Porcine Proliferative Enteropathy(PPE). At regular times before and after challenge serum blood andfaeces samples were taken from the pigs for serology and PCRrespectively. At 68 days of age all pigs were euthanized and post-mortemexamined. The ileum was examined histologically.

The other issues in the experimental design were the same as describedin Example 2, unless indicated otherwise.

Results of Example 3

Serology

Before first vaccination all pigs were seronegative for IFT Lawsoniaantibody titres. After vaccination with the combi vaccine (groups 1 and2) and the non-protein carbohydrate vaccine (group 4), many pigsdeveloped IFT antibody titres whereas the controls and the pigsvaccinated with Enterisol remained seronegative until challenge. Afterchallenge all pigs (except two in the Enterisol group) developed IFTantibody titres. For an overview of the mean values obtained, see table4 (due to the higher dilution when compared to example 2, the detectionlevel was 4.0).

TABLE 4 Mean IFT Lawsonia antibody titres (2log) of pig serum aftervaccination and challenge Group T = 3 days T = 25 days T = 46 days T =67 days 1 <4.0 <4.0 7.9 10.3 2 <4.0 <4.0 4.8 9.8 3 <4.0 <4.0 <4.0 8.5 4<4.0 <4.0 6.9 10.6 5 <4.0 <4.0 <4.0 9.0

With respect to Mhyo, at the start of the experiment as well as day ofbooster (25-day-old) all pigs were seronegative for Mhyo. After boostervaccination group 1 developed high Mhyo antibody titres, comparable tothose obtained with the commercially available vaccine.

With respect to PCV, at 3-day-old the piglets had high maternallyderived PCV antibody titres. At day of booster (25-day-old) thevaccinates (group 1) had a similar titre compared to group 2 and thecontrol group. The PCV titre at 25-day-old was slightly lower comparedto the titre at 3-day-old. After the vaccination at 25-day-old thetitres of group 1 (2 vaccinations at day 3 and 25) and group 2 (onevaccination at day 25) remained at a high level whereas control pigletsshowed a normal decrease in maternally derived antibodies. The PCVtitres obtained are comparable to the titres obtainable withcommercially available vaccines.

Real-Time PCR on Faeces Samples

Three weeks after challenge, pigs of group 1, 2 and 4 had less Lawsonia(DNA) in their feces compared to groups 3 and 5. Only the differencesbetween group 1 and 3 (Enterisol) and group 4 and 3 were statisticallysignificant (p<0.05, Mann-Whitney U test). For the mean results, seetable 5.

TABLE 5 Mean results of PCR on faeces samples (log pg) after vaccinationand challenge Group Mean value 1 1.0 2 1.2 3 2.0 4 0.6 5 1.8

Histological Scores

Histology scores of group 1 and 4 were significantly lower compared tothose of groups 3 and 5 (p<0.05, two-sided Mann-Whitney U test (seetable 6). The number of pigs with confirmed PPE were 2/13 in group 1,6/12 in group 2, 12/14 in group 3, 2/7 in group 4 and 12/14 in thecontrol group 5. Groups 1 and 4 had a significantly lower incidence ofPPE compared to groups 3 and 5 (p<0.05, two-sided Fischers' exact test).

TABLE 6 Mean histology score for the ileum. Group HE Score IHC ScoreTotal Score 1 0.4 0.6 1.0 2 0.7 0.7 1.4 3 1.6 1.4 3.0 4 0.4 0.4 0.8 51.9 1.5 3.4

Conclusion of Example 3

From the results it can be concluded that systemic administration of thewhole cell Lawsonia bacterin combined with PCV and Mhyo antigen as wellas the vaccine comprising (substantially protein-free) the carbohydrate,administered at 3-day-old and 25-day-old, both induce partial protectionagainst experimental Lawsonia intracellularis infection. It isparticularly surprising that the vaccine is effective when the primeadministration takes place before weaning (younger than 21-25 days). Itis noted that in the examples 2 and 3 the vaccines as far as Lawsoniaantigens are concerned, per ml contain antigenic material derived frommore than 1E8 Lawsonia intracellucaris cells. Given the fact that thesevaccines, even though mild adjuvants are being used (viz. adjuvantscontaining small droplets and no or little mineral oil), confer goodprotection against ileitis, in particular when compared to thecommercially available vaccine Enterisol® Ileitis, the dose of antigenscould be lowered. This could be done by administering less vaccine (downto e.g. 0.2 ml, suitable for e.g. intradermal application), ordecreasing the antigenic content of the vaccine. Based on analogues invaccine technology it is believed that with an antigenic dose (pervaccination) derived from or containing 1E7 cells, in particular 2.5E7cells or higher, still comparable or even better results can be obtainedthan with the current commercially available vaccine. Given the factthat the combination vaccine provided titres for Mhyo and PCV antibodiesto a level comparable with the levels obtainable with commerciallyavailable single vaccines, it is understood that the combination vaccinealso provides protection against Mycoplasma hyopneumoniae and Porcinecirco virus.

EXAMPLE 4

This experiment was conducted to establish a dosage affect of a vaccineaccording to the invention. Also in this experiment unvaccinated animalswere used as a control.

Experimental Design of Example 4

Inactivated whole cell vaccines were made as indicated in example 2. Theantigenic material was formulated in Diluvac forte adjuvant at aconcentration of approximately 2.0×10⁸ cells per ml vaccine,respectively 5.0×10⁷ and 1.25×10⁷ cells per ml vaccine. Sixty 3-day-oldSPF piglets were used. The pigs were allotted to four groups of 15 pigseach. The piglets of groups 1, 2 and 3 were vaccinated intramuscularly(in the neck) at 3-day old and 25-day-old with 2 ml of the vaccine eachtime. Group 4 was left as unvaccinated control. At 46-day-old all pigswere challenged orally with Lawsonia bacteria as indicated under example2. At 67-day-old all pigs were euthanized and examined. Tests wereperformed as indicated under example 2. Next to this PCR was peformed onmucosa samples. For this, an ileum sample was taken from every animal,where applicable from an area which showed thickening.

Results of Example 4

Weight Gain

From 14 days and onwards significant differences in total weight gainappeared among the groups. Group 1 showed an average total weight gainof approximately 5350 grams. In group 2 this was 5150 grams. Group 3showed a weight gain of about 4250 grams, whereas Group 4 showed aweight gain of 4550 grams.

Real-Time PCR on Faeces Samples

Three weeks after challenge positive reactions were found in all groups.Group 1 and Group 2 had a significantly lower shedding level compared tothe control. A post-challenge overview of the number of infected animals(as determined by PCR) is given in table 7.

TABLE 7 Result of PCR on faeces samples after vaccination and challengeGroup Number of infected animals post-challenge 1 1/15 2 2/15 3 7/15 48/15

Real-Time PCR on Mucosa Samples

Three weeks after challenge positive reactions were found in all groups.Group 1 and Group 2 had a significantly lower shedding level compared tothe control. A post-challenge overview of the number of infected animals(as determined by PCR) is given in table 8.

TABLE 8 Result of PCR on mucosa samples after vaccination and challengeGroup Number of infected animals post-challenge 1 0/15 2 2/15 3 5/15 46/14 (no sample of pig no 8)

Histology Scores

The total histology score and the number of animals which were confirmedto have PPE are depicted in table 9.

TABLE 9 Mean histology score for the ileum. Group Total score Number ofanimals with PPE 1 0.3 0/15 2 0.8 2/14 3 1.1 4/15 4 1.9 7/15

Conclusion of Example 4

Contrary to what was expected, the results indicate that there is a verysudden decrease in protective effect around the lowest dosage used inthese experiments. Although a dosage of antigenic material derived from2.5×10⁷ cells still provided a protective effect comparable with that ofthe commercially available vaccine, the fact the decrease between adosage that is only 0.6 log higher is so significant (hardly any effectseen in weight gain, number of infected animals and PCR on mucosa;however, still a decrease in number of PPE recognized animals), gave theinsight that in general a practical lowest effective dose of the antigencan be derived at: an amount of antigens less than derived from orcontaining 1×10⁷ cells will in practice, under the current marketcircumstances, not lead to economically relevant results. The reason forthe existence of this apparent cut-off value is not 100% clear. Usuallyone expects a more gradual decrease in protection when the dosage islowered. It might be that combating a local infection in the mucosa ofthe intestines via a systemically derived immune response needs aminimum amount of antigens.

Next to the above, the surprising effect seen in Example 3, viz. that avaccine based on a carbohydrate antigen administered systemically, iseffective when the prime administration takes place before weaning(younger than 21-25 days), is confirmed in this experiment with the useof another adjuvant. Therefore it can reasonably be understood that thisfeature is generic for a non-live vaccine comprising a carbohydrateantigen.

The invention claimed is:
 1. A method of immunizing a pig againstLawsonia intracellularis comprising systemically administering to thepig a single dose of a composition comprising non-live inactivated wholecell Lawsonia intracellularis bacteria at a concentration of more thanor equal to 5.0×10⁷ cells per ml.
 2. The method according to claim 1,wherein the composition further comprises an oil in water adjuvantcontaining oil droplets of sub-micrometer size.
 3. The method accordingto claim 2, wherein the adjuvant comprises droplets of biodegradable oiland droplets of mineral oil, the droplets of biodegradable oil having anaverage size that differs from the average size of the droplets ofmineral oil.